PROJECT SUMMARY Although Roux-en-Y Gastric Bypass (RYGB) is one of the most effective treatments for obesity and type 2 Diabetes Mellitus (T2DM), one out of three patients, still suffer from T2DM after the operation. Identifying those patients who do not achieve T2DM remission is important both mechanistically to help discover how RYGB improves glycemia, and clinically to initiate additional postoperative treatments early after surgery or to avoid the operation altogether. Studies in animal models and humans have emphasized the role of the intestine as a key anatomic substrate of the mechanisms of metabolic improvement after RYGB. The proposed project builds upon and expands the findings of our recent, prospective, longitudinal, observational clinical trial (ClinicalTrials.gov ID NCT02710370) the goal of which was to characterize the nature and the timing of RYGB- induced intestinal adaptive changes in humans. The three main implications that emerged from this trial were a) the enhancement of intestinal fuel utilization contributes in a significant and quantifiable manner to the 1- year postoperative decrement in HbA1c, positioning the gut as a key organ for RYGB-induced improved glycemia, b) the preoperative intestinal biology appears to be correlated with postoperative response, and thus this could be harnessed as a predictive clinical tool, and c) SIRT6-HIF1A pathway, a master gatekeeper of glucose metabolism linking energy substrate metabolism to function, appears to play a prominent role in the glycemic control of RYGB. The proposed project is a unique synergy between basic and clinical research teams and contains the requisite sophistication to further evaluate the role of augmented intestinal metabolism as one of the key mechanisms of action of RYGB and as a clinical tool to predict T2DM remission. We will perform 3 studies on human subjects. In Specific Aim 1, we will conduct a case control study to test the hypothesis that patients without T2DM remission post-RYGB exhibit a lower degree of intestinal metabolic activity and lower ability to engage SIRT6-HIF1A pathway effectively. In Specific Aim 2, we will conduct a prospective, longitudinal study to test whether specific composite metrics that take into account the preoperative gene expression levels could help predict postoperative HbA1c change, alone or in combination with other clinical, metabolic or hormonal factors. In Specific Aim 3, we will perform single cell RNA- Sequencing in biopsies from subjects before and after RYGB. These studies will further clarify the role of intestinal metabolism and the SIRT6-HIF1A pathway in RYGB-induced glycemic control. They will allow us to gain unprecedented insight into the intestinal cellular substrates, the regulatory networks and the mechanisms that are relevant for T2DM remission. They may also, potentially pave the way for the development of molecular predictors that could help guide clinical decisions for bariatric surgery candidates. Finally, they will generate unique resources, biobanks and datasets that will further enable mechanistic studies of intestinal biology and the mechanisms of action of bariatric surgery unobtainable to date.